Characterization of the chondrocyte secretome in photoclickable poly(ethylene glycol) hydrogels

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Abstract

<p class="first" id="P1">Poly(ethylene glycol) (PEG) hydrogels are highly tunable platforms that are promising cell delivery vehicles for chondrocytes and cartilage tissue engineering. In addition to characterizing the type of extracellular matrix (ECM) that forms, understanding the types of proteins that are secreted by encapsulated cells may be important. Thus, the objectives for this study were to characterize the secretome of chondrocytes encapsulated in PEG hydrogels and determine whether the secretome varies as a function of hydrogel stiffness and culture condition. Bovine chondrocytes were encapsulated in photoclickable PEG hydrogels with a compressive modulus of 8 and 46 kPa and cultured under free swelling or dynamic compressive loading conditions. Cartilage ECM deposition was assessed by biochemical assays and immunohistochemistry. The conditioned medium was analyzed by liquid chromatography-tandem mass spectrometry. Chondrocytes maintained their phenotype within the hydrogels and deposited cartilage-specific ECM that increased over time and included aggrecan and collagens II and VI. Analysis of the secretome revealed a total of 64 proteins, which were largely similar among all experimental conditions. The identified proteins have diverse functions such as biological regulation, response to stress, and collagen fibril organization. Notably, many of the proteins important to the assembly of a collagen-rich cartilage ECM were identified and included collagen types II(α1), VI (α1, α2, and α3), IX (α1), XI (α1 and α2) and biglycan. In addition, many of the other identified proteins have been reported to be present within cell-secreted exosomes. In summary, chondrocytes encapsulated within photoclickable PEG hydrogels secrete many types of proteins that diffuse out of the hydrogel and which have diverse functions, but which are largely preserved across different hydrogel culture environments. </p><p id="P2"> <div class="figure-container so-text-align-c"> <img alt="" class="figure" src="/document_file/24467fce-5ebc-49ff-92db-6a81c497614a/PubMedCentral/image/nihms872833u1.jpg"/> </div> </p>

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Charles Hoyle, Christopher Bowman (2010)

Following Sharpless' visionary characterization of several idealized reactions as click reactions, the materials science and synthetic chemistry communities have pursued numerous routes toward the identification and implementation of these click reactions. Herein, we review the radical-mediated thiol-ene reaction as one such click reaction. This reaction has all the desirable features of a click reaction, being highly efficient, simple to execute with no side products and proceeding rapidly to high yield. Further, the thiol-ene reaction is most frequently photoinitiated, particularly for photopolymerizations resulting in highly uniform polymer networks, promoting unique capabilities related to spatial and temporal control of the click reaction. The reaction mechanism and its implementation in various synthetic methodologies, biofunctionalization, surface and polymer modification, and polymerization are all reviewed.

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Garret D Nicodemus, Stephanie J Bryant (2008)

Encapsulating cells in biodegradable hydrogels offers numerous attractive features for tissue engineering, including ease of handling, a highly hydrated tissue-like environment for cell and tissue growth, and the ability to form in vivo. Many properties important to the design of a hydrogel scaffold, such as swelling, mechanical properties, degradation, and diffusion, are closely linked to the crosslinked structure of the hydrogel, which is controlled through a variety of different processing conditions. Degradation may be tuned by incorporating hydrolytically or enzymatically labile segments into the hydrogel or by using natural biopolymers that are susceptible to enzymatic degradation. Because cells are present during the gelation process, the number of suitable chemistries and formulations are limited. In this review, we describe important considerations for designing biodegradable hydrogels for cell encapsulation and highlight recent advances in material design and their applications in tissue engineering.

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Jennifer S Park, Julia S F Chu, Anchi D. Tsou … (2011)

Bone marrow mesenchymal stem cells (MSCs) are a valuable cell source for tissue engineering and regenerative medicine. Transforming growth factor β (TGF-β) can promote MSC differentiation into either smooth muscle cells (SMCs) or chondrogenic cells. Here we showed that the stiffness of cell adhesion substrates modulated these differential effects. MSCs on soft substrates had less spreading, fewer stress fibers and lower proliferation rate than MSCs on stiff substrates. MSCs on stiff substrates had higher expression of SMC markers α-actin and calponin-1; in contrast, MSCs on soft substrates had a higher expression of chondrogenic marker collagen-II and adipogenic marker lipoprotein lipase (LPL). TGF-β increased SMC marker expression on stiff substrates. However, TGF-β increased chondrogenic marker expression and suppressed adipogenic marker expression on soft substrates, while adipogenic medium and soft substrates induced adipogenic differentiation effectively. Rho GTPase was involved in the expression of all aforementioned lineage markers, but did not account for the differential effects of substrate stiffness. In addition, soft substrates did not significantly affect Rho activity, but inhibited Rho-induced stress fiber formation and α-actin assembly. Further analysis showed that MSCs on soft substrates had weaker cell adhesion, and that the suppression of cell adhesion strength mimicked the effects of soft substrates on the lineage marker expression. These results provide insights of how substrate stiffness differentially regulates stem cell differentiation, and have significant implications for the design of biomaterials with appropriate mechanical property for tissue regeneration. Copyright © 2011 Elsevier Ltd. All rights reserved.